characterisation of volatile constituents of six native

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Characterisation of volatile constituents of six native New Zealand ferns and changes in volatile emission in response to

herbivore, mechanical wounding and phytohormone treatments.

A thesis presented in partial fulfilment of the

requirements for the degree of

Master of Science

in

Ecology

At Massey University, Manawatū Campus, New Zealand

Keylee Soriano

2018

i

Abstract

Evolution has led to the development of countless defence strategies in terrestrial plants to deal

with the threat of herbivory and disease. The production of specialised morphological

structures, such as thorns and trichomes, is a prominent defence mechanism that directly deters

potential herbivores. However, plants are capable of enlisting the aid of natural predators and

parasites of attacking herbivores as a means of indirect defence, through the production and

release of volatile organic compounds. This has prompted much research into the regulation

and ecological roles of volatile organic compounds in many higher plant groups. However,

similar studies are seldom in lower plants such as the Monilophytes thus we know very little

concerning the ecological importance of plant emitted volatiles in this group. In this study, I

investigated the volatile compounds released by six abundant native fern species using direct

solvent extraction and headspace collections, and characterized the volatile emissions under

natural herbivory, phytohormone treatment, and mechanically induced stress. Solvent extracts

and headspace collections were analysed using gas chromatography coupled with mass

spectrometry allowing the quantitative and qualitative description of the volatile profiles. These

results were then used to relate volatile emission to the growth mode and other potential

defence strategies of these species. A total of 15 volatile compounds were identified over the

course of this thesis with links to fern physiology. Quantitative results revealed no differences

in emissions under phytohormone treatment or artificially induced stress. The comparison of

two methods, solvent extraction and headspace sampling, reveals the limitations the solvent

extraction method has on elucidating fern-insect interactions. Research on fern volatiles could

give insight into the evolution of anti-herbivore defence mechanisms in plants and the

interactions between native ferns and arthropod communities. Potential applications of

research in fern chemistry include pharmaceutical or perfumery uses and fern conservation,

which should be incentives for further work. The results and conclusions made from this thesis

does not only contribute to the limited pool of knowledge in this field of research but may also

serve as a foundation for future studies.

iii

Acknowledgements

This thesis would not have been possible without the support and input of several people. I

would like to express, first and foremost, my gratitude and appreciation to my supervisor Dr.

Andrea McCormick who not only introduced me to this amazing field of research but also

encouraged and advised me all throughout my Masters. A huge thank you for proof-reading and

providing feedback over the course of writing my thesis, your enthusiasm in particular, in

addition to your expertise in this subject were the driving forces behind me completing this

dissertation. Despite your ever-busy schedule, you always managed to make time for me and

others whenever your door was open which I, and very likely the others as well, will always be

grateful for. Those of whom I also owe a great deal of thanks for their contribution are Paul

Barrett, the technical manager for the Ecology department, for not only procuring the

equipment I needed for my experiments but also swiftly resupplying consumables whenever

they were exhausted. John Sykes, primary technician for the Environmental Engineering

Laboratory, for allowing me to use their laboratory’s GC-MS and for also maintaining and

servicing the machine whenever it occasionally misbehaved. Shaun Nielsen, an ecology

technician, for his advice in my herbivory experiments and his willingness to get dirty and muddy

for the sake of catching my wetas, I cannot thank you enough for this. Dr. Gillian Rapson, a senior

lecturer in Ecology, who graciously lent me some space for my ferns in her nursery. I would like

to give my gratitude to the entire Chemical Ecology group, past and present, of which I am apart

of for their continuous support, encouragement and assistance throughout my studies. I would

like to acknowledge Massey University overall for providing the services needed to accomplish

my Masters studies. Lastly, I would like to express my thanks to my family, my parents in

particular, for supporting me all through my tertiary education be it financially or emotionally. I

could not have made it without you. Looking back on the past several years it has certainly been

quite an enjoyable experience, I have learnt many things and have grown and matured as a

person. To everyone, both directly and indirectly, involved in my journey at Massey University

it has been an absolute pleasure working and interacting with you all and I look forward to what

comes next.

iv

Table of Contents Abstract ....................................................................................................................................... i

Acknowledgements ................................................................................................................... iii

Table of Contents ...................................................................................................................... iv

List of Figures ............................................................................................................................ vi

List of Tables ............................................................................................................................ vii

List of Abbreviations ............................................................................................................... viii

Chapter 1 : General Introduction and Literature Review ........................................................... 1

Plant Secondary Metabolites .................................................................................................... 3

Role and Importance ................................................................................................................. 4

Classification ............................................................................................................................. 5

Plant Volatiles and their Ecological Roles ................................................................................. 7

Secondary Compounds in Ferns ................................................................................................ 9

Fern Insect Interactions .......................................................................................................... 11

Methods for Volatile Collection and Analysis ......................................................................... 12

Research Objectives ................................................................................................................ 14

Chapter 2 : Materials and Methods ........................................................................................... 17

Study Site and Fern Species .................................................................................................... 19

Solvent Extraction ................................................................................................................... 20

Phytohormone and Mechanical Damage ............................................................................... 21

Polyphenol & Chlorophyll Collection ...................................................................................... 23

Herbivore Damage .................................................................................................................. 23

Gas Chromatography-Mass Spectre Analysis ......................................................................... 26

Statistical Analyses .................................................................................................................. 28

Chapter 3 : Results...................................................................................................................... 31

Solvent Extraction ................................................................................................................... 33

Phytohormone and Mechanical Damage ................................... Error! Bookmark not defined.

Polyphenol and Chlorophyll Analysis ...................................................................................... 41


v

Chapter 4 : General Discussion .................................................................................................. 47

Solvent Extraction vs. Headspace Collection .......................................................................... 49

Phytohormone Treatment and Mechanical Damage ............................................................. 50

Polyphenol and Chlorophyll Analyses ..................................................................................... 53


Final Remarks .......................................................................................................................... 55

Chapter 5 : Conclusions and Recommendations ....................................................................... 59

Conclusions ............................................................................................................................. 61

Recommendations for Future Work ....................................................................................... 62

Literature Cited ........................................................................................................................... 65

vi

List of Figures

Figure 2-1. The location of vegetative areas (red circles) within Massey University (Google Earth). .......................................................................................................................................... 19 Figure 2-2. Native fern species used for volatile analysis; (a) Dicksonia squarrosa, (b) Microsorum pustulatum, (c) Asplenium bulbiferum, (d) Asplenium oblongifolium, (e) Cyathea dealbata, (f) Cyathea medullaris. ................................................................................................ 20 Figure 2-3. Examples of solvent extraction process: (a) initial filtering process using 70mm glass microfiber filter, (b) sealed containers containing fern material submerged in solvent/internal standard solution. ....................................................................................................................... 21 Figure 2-4. Example of a field experimental setup of the dynamic volatile headspace collection system using a PVAS22 unit on Dicksonia squarrosa. ................................................................. 22 Figure 2-5. Example of herbivore damage by H. crassidens on frond cuttings of C. medullaris in preliminary trials. ........................................................................................................................ 23 Figure 2-6. Example of a laboratory experimental setup of the dynamic volatile headspace collection system using a PVAS22 unit on D. squarrosa. ............................................................ 24 Figure 2-7. Examples of insects interacting with ferns: (a) S. australis resting on D. squarrosa, (b) H. crassidens consuming pinna leaves of D. squarrosa. ........................................................ 25 Figure 2-8. Example of manual peak integration using ion chromatography. ........................... 26 Figure 2-9. Example of compound identification comparing mass spectrum data from samples to target reference values within the NIST05 MS library. .......................................................... 27 Figure 3-1. Comparisons of confidence intervals (95%) and mean values of the concentrations of fern extracts according to species (a) and method of growth (b). ......................................... 33 Figure 3-2. Comparison of total volatile emission rates for each treatment; control - no induced damage (C) and two treatments - mechanical damage (PD), and phytohormone treatment (JA). .................................................................................................................................................... 35 Figure 3-3. Comparison of total volatile emission rates for each treatment according to fern species. Control (CT) with two treatments - mechanical damage (PD), and phytohormone treatment (JA). ............................................................................................................................ 39 Figure 3-4. Comparison of total volatile emission rates for each treatment according to fern growth mode. Control (CT) with two treatments - mechanical damage (PD), and phytohormone treatment (JA). (Note: Tree = CD, CM and DS; Epiphyte = MP; Shrub = AB and AO). ................ 40 Figure 3-5. Comparisons of confidence intervals (95%) and mean values of chlorophyll content (a); flavonols (b); anthocyanins (c); and nitrogen balance index (d) of the six native fern species. ........................................................................................................................................ 41 Figure 3-6. Comparisons of confidence intervals (95%) and mean values of chlorophyll content (a); flavonols (b); anthocyanins (c); and nitrogen balance index (d) of the three growth modes. (Note: Tree = CD, CM and DS; Epiphyte = MP; Shrub = AB and AO). .......................................... 42 Figure 3-7. Example of three ion chromatograms of a control replicate for A. bulbiferum. (a) Chromatogram of volatile measurements prior to herbivore application period, (b) Chromatogram of volatile measurements during the herbivore application period, (c) Chromatogram of volatile measurements 14 hours after herbivore application period. Note: Red lines indicate target volatile compounds. ............................................................................ 44

vii

List of Tables

Table 3-1. Average concentration of fern extract compounds from fronds of six species. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 FW. Note: n.d – no data available. ..................................................................................................... 34 Table 3-2. Average emission and SE of volatile organic compounds from fronds of six fern species within the control treatment. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 DW h-1. Note: n.d – no data available. ....................... 36 Table 3-3. Average emission and SE of volatile organic compounds from fronds of six fern species within the phytohormone (jasmonic acid) treatment. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 DW h-1. Note: n.d – no data available. ............................................................................................................................. 37 Table 3-4. Average emission and SE of volatile organic compounds from fronds of six fern species within the mechanical damage only treatment. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 DW h-1. Note: n.d – no data available. ..................................................................................................................................... 38 Table 3-5. List of compounds detected from the herbivory experiments and corresponding treatments they were observed within. Control (CT), Weta (W) - H. crassidens, Passion Vine Hopper (PVH) – S. australis. n.d – no data available. .................................................................. 43

viii

List of Abbreviations

VOC - Volatile Organic Compounds

CT - Control

JA - Jasmonic Acid

PD - Mechanical Damage

W – Weta

PVH - Passion Vine Hopper

GC-MS - Gas Chromatography–Mass Spectrometry

AB - Asplenium bulbiferum

AO - Asplenium oblongifolium

CD - Cyathea dealbata

CM - Cyathea medullaris

DS - Dicksonia squarrosa

MP - Microsorum pustulatum

Chapter 1 : General Introduction and Literature Review

3

Plant Secondary Metabolites

Metabolites are the products and intermediates of metabolism, a process cells within all living

organisms undergo, and is usually restricted to small molecules. Metabolites have various

functions including the conversion of food into energy, stimulation/inhibition of enzymes and

defence (Demain & Fang, 2000; Vining, 1990). They also play roles in interactions between

other organisms such as their importance in the production of odours and pheromones (Hook

et al., 1991; Stacey, 2003). Countless years of modern biochemistry have separated

metabolites into two separate categories, primary and secondary, according to their

involvement in the development of living cells. Primary metabolites are described as having

roles in basic life functions such as growth, respiration, cell division and reproduction, whereas

secondary metabolites are not directly involved in these processes but usually serve important

ecological functions (Bourgaud et al., 2001).

The concept of secondary metabolites can be attributed to Stahl (1888) who proposed the

name ‘Schutzexkrete’ for essential oils, alkaloids, tannins and other secondary metabolites.

Kossel (1891) then defined these as separate to primary ones. It was not until research by

Czapek (1921) who dedicated a volume of his ‘plant biochemistry’ series to what he called

‘endproduckt’, that secondary metabolites became widely recognized.

Unlike the main molecules found in plants, secondary metabolites were then characteristically

defined by their low abundance - less than 1% of total carbon occurring in organs or dedicated

cells (Rao & Ravishankar, 2002). Through the rapid improvement and development of

analytical techniques, such as chromatography, more and more of these molecules could be

recovered and as a result, phytochemistry established as a discipline during the 20th century.

Although paper chromatography initially identified some molecules to be pigments, other

functions of these secondary compounds within plants remained unknown. However, with the

continuous improvement of biochemical techniques and the birth of modern molecular

biology, these secondary products have been shown to evidently play a role in how plants

adjust and interact with their environment (Bourgaud et al., 2001). Secondary metabolites

typically arise as offshoots from the pathways of primary metabolism (Geissman & Crout,

1969). They can be classified according to their composition (e.g. contain nitrogen), chemical

structure (e.g. rings) but usually they are classified according to their biosynthetic pathways

(Rosenthal & Berenbaum, 2012).

4

A few words of clarification must be said before we proceed, the term ‘secondary metabolites’

is rather misleading and may cause many misunderstandings amongst those unfamiliar with

chemical ecology. This term is rather vague to begin with as it indiscriminately covers a very

wide range of unrelated compounds and implies an unimportant, ‘secondary’ role for them.

As we begin to increase our understanding and knowledge regarding secondary metabolites,

the previously held belief that they were merely just functionless molecules or waste products

resulting from ‘mistakes’ of primary metabolism has become a largely inaccurate and

misguided view of these compounds. Rather many of these ‘secondary’ products are key

components of active and potent defence mechanisms and are also significant components in

signalling molecules (Robbins, 2000).

Although this way of thinking is relatively new and has only recently gained traction amongst

the scientific community, many have come to the consensus that these products are part of a

long-standing evolutionary arms race in a sort of ‘chemical warfare’ fought between plants and

their pests and pathogens as proposed several years earlier by Bennett and Wallsgrove (1994).

Role and Importance

In higher plants, secondary metabolites contribute to the specific tastes, odours and colours

observed in plants (Bennett & Wallsgrove, 1994), and are crucial to the human industry as they

are unique sources for pharmaceutical products, food additives, cosmetics and multiple other

products (Ravishankar & Rao, 2000). In the environment, these compounds are key to plant

survival and many have several important ecological functions. These functions can be

condensed into three distinct roles: 1) They serve as attractants for pollinators, seed-dispersing

animals and herbivores (Cipollini & Levey, 1997; Kessler & Baldwin, 2007); 2) Protect plants

against biotic and abiotic stressors (Bartwal et al., 2013; Edreva et al., 2008); and 3) Function

as agents of plant-microbe symbiosis and mediate plant-plant communication (Bais et al.,

2004; Reichling, 2010).

By nature, plants are immobile organisms vulnerable to their surrounding environment and

must therefore adapt in order to survive. To compensate for their inability to flee from or fight

against incoming threats, plants have evolved unique defence mechanisms to cope with

herbivores and pathogens. These mechanisms are often divided into direct and indirect

defences. Direct plant defences (direct resistance) refer to traits that act upon the attacker

directly (Gols, 2014) and include the production of specialized morphological structures (e.g.

thorns, spines, thicker leaves, trichomes etc.) and chemical substances (e.g. alkaloids,

5

phenolics, cyanogenic glucosides, etc.) (Chen, 2008) to discourage herbivores from causing

physical damage or eliciting physiological reactions in the plant (War et al., 2012).

In contrast, indirect defences promote the efficiency of natural enemies to control plant

antagonists (Heil, 2008) and include the release of volatile organic compounds (VOC’s) from

flowers, leaves and fruits into the immediate atmosphere (Maffei, 2010) as well as from the

roots (Rasmann et al., 2005) into the soil to attract the natural enemies of their attackers

(Dicke & Sabelis, 1987).

Classification

Based on their biosynthetic origins, plant secondary metabolites can be divided into three

major groups: phenolics, terpenoids and nitrogen-containing compounds. Each group is

chemically distinct from one another and are unique components of defence mechanisms in

plants (Bennett & Wallsgrove, 1994). However, that is not to say that other minor chemical

groups are less important to plant physiology and fitness but rather, the diversity and

broadness of these classes would be of more use to analyse when there is much left yet to

understand regarding fern physiology.

Terpenes (terpenoids), make up the largest class of secondary metabolites and are generally

insoluble in water as they consist mostly of resins and essential oils (Singh & Sharma, 2015).

Although the vast majority of terpenes are secondary compounds likely involved in plant

defences, there are a select few compounds directly involved in plant growth or development

and as such can be considered primary rather than secondary metabolites (e.g. gibberellins,

brassinosteroids) (Campbell & Reece, 2002). Terpenes have been shown to be key factors in

the resistance of several pathogens and insect pests (Harborne, 2014). For example, in

conifers, monoterpenes accumulate in resin ducts found in the trunk, needles and twigs

(Franceschi et al., 2005) and are toxic to the bark-beetle species that infest these plants.

However, this is entirely dependent on the conifer and beetle species in question, observations

of these monoterpenes across related species show that it can act as both a repellent and

attractant of herbivore pests suggesting a co-evolutionary arms race between the beetles and

plant.

Terpenes are also prime examples of how plants indirectly defend themselves against

herbivores via enhancing the effectiveness of the natural enemies of the herbivores. Research

on cotton plants by Rodriguez-Saona et al. (2003) demonstrate that, in the absence of

6

competing herbivores, natural enemies respond to both the volatile and non-volatile emissions

of terpenoid compounds emitted by the beet armyworm through herbivory.

Phenolic compounds are appropriately defined by their characteristic phenol group: a hydroxyl

functional group on an aromatic ring. Plant phenolics are a ubiquitous, chemically

heterogeneous group with a majority of compounds expressing a diverse range of chemical

properties (Hopkins & Huner, 2009).

Like terpenes, phenolics function both to attract and repel different organisms within the

plants surroundings. Phenols are capable of acting as inhibitors, protective agents, pesticides

and natural animal toxicants against attacking organisms (such as herbivores and

bacterial/fungal pathogens) (Bhattacharya et al., 2010). Examples of phenolics acting as

inhibitors of enzymes and feeding deterrents of herbivores are documented (Cheeke, 1989),

however evidence in their role in resistance against bacteria and fungi pathogens are more

pronounced (Bennett & Wallsgrove, 1994). For instance, the production of certain phytoalexin

(anti-microbial) compounds (polyphenols) against the growth of fungal pathogens in two

grapevine species (Favaron et al., 2009; Timperio et al., 2012) emphasizes the dynamic role

which phenolic compounds possess in the deterrence of microbial organisms compared with

that of plant-herbivore stress.

Phenolics are also recognised as allelochemicals for competitive plants and weeds (Xuan et al.,

2005). Some phenolic metabolites (e.g. salicylic acid) are phytotoxic and can accumulate in the

soil and subsequently inhibit seedling growth and seed germination of nearby plants (Baleroni

et al., 2000).

Nitrogen-containing compounds are as common as the previous classes of metabolites.

Alkaloids and cyanogenic glycosides in particular are of significant interest not only due to their

effectiveness as anti-herbivore defences (Schardl & Chen, 2010) but also because of their

medicinal properties and toxicity to humans. These compounds are mostly products of

common amino acids (Lazar et al., 2003). It was initially assumed that alkaloids were

nitrogenous wastes (i.e. urea and uric acid) however there was very little evidence in support

of these hypotheses and ultimately refuted (Meyers, 1987). However, although some believe it

to function as defence against herbivores due to their toxicity and general deterrence

capability (Hopkins & Huner, 2009) many have yet to agree since there are areas concerning

alkaloids left unclear (Aniszewski, 2015).

7

It has long been known that alkaloids have some effect on herbivorous insects, studies dating

back almost four decades (Hesse, 2002) have demonstrated that alkaloids do play a role in

plant-insect resistance. For example, increasing concentrations of alkaloids by Solanum (potato

genus) were positively correlated with reduction in leaf hopper infestation, a common pest

(Sanford et al., 1990). Similarly, in barley, concentrations of the alkaloid gramine decreased as

the plant matured, this decrease in alkaloid concertation was directly correlated with increases

in the rate of survival and feeding of aphid pests suggesting a deterrence function of gramine

in barely seedlings (Zúñiga et al., 1986).

Plant Volatiles and their Ecological Roles

As discussed in the previous section, plants can produce a wide array of metabolic compounds

some of which are volatile. There is substantial evidence showing that higher plants are

capable of producing a large variety of volatile compounds (Knudsen et al., 1993). Volatile

compounds generally consist of lipophilic substances with high vapour pressures capable of

crossing membranes freely in the absence of a diffusion barrier (Pichersky et al., 2006). Plant

volatiles are usually complex mixtures of the chemical compounds produced by the plant

(Baldwin, 2010) and the composition and intensity of the volatile blends can carry vital

information concerning the physiological status of the emitting plant and the biotic and abiotic

stresses it is experiencing (Holopainen & Gershenzon, 2010; Vickers et al. 2009).

The availability of elements such as carbon, sulphur and nitrogen in addition to the energy

provided through primary metabolism dictate the biosynthesis of volatile organic compounds

(VOC’s) within the plant (Vallarino et al. 2018). Depending on the chemical structure of these

compounds, plant volatiles can be classed into alcohols, aldehydes, esters, ethers,

hydrocarbons and ketones. However, finer classifications of these volatiles can also be done

based on their biosynthetic origin and are generally divided into terpenoids, fatty acid

derivatives, amino acid derivatives and benzenoids/phenylpropanoids.

Terpenoids (isoprenoids) and other steroids are products of three biosynthetic pathways;

mevalonate, methylerythritol phosphate and Deoxyxylulose phosphate (Eisenreich et al.,

2001), fatty acids and polyketides come from the acetate pathway, and aromatic amino acids

and phenylpropanoids originate from the shikimate (shikimic acid) pathway (Dewick, 2002;

Ribera & Zúñiga, 2012; Tzin & Galili, 2010).

8

Although these compounds are generally found in relatively small amounts when compared to

the total weight of the plant, collectively they possess important ecological functions

(Rosenkranz & Schnitzler, 2016). They are primarily associated with plant indirect and direct

defences by either attracting the natural enemies of herbivores (Paré & Tumlinson, 1999) or by

acting as feeding and/or oviposition deterrents (Arimura et al., 2009). For example, many

volatile terpenes emitted by plants are non-specific toxins which are active against a range of

organisms (e.g. bacteria, animals) (Rosenkranz & Schnitzler, 2016).

Plant-emitted volatiles mediate communication between plants and other organisms. Seed

dispersers and pollinators are attracted by volatile compounds thus increasing the

reproductive advantage of the plant (Mumm & Dicke, 2010; Pichersky & Gershenzon, 2002).

Ironically, as a product of coevolution, certain phytophagous insects are also capable of using

volatiles to locate suitable host plants and mates (Landolt & Philips, 1997; Reddy & Guerrero,

2004) yet, in most cases, plant volatiles primarily function as an indirect defence mechanism

against them.

Changes in the emission of VOC’s as a result of herbivore damage attract natural enemies

(predators and parasites) of feeding herbivores to the plant (Turlings et al., 1995). Depending

on the type of stress and the plant species involved, quantitative and qualitative differences

between volatile bouquets help herbivore enemies discriminate between status of host plants

(Olson et al., 2009) and herbivore species (De Boer et al., 2004; De Moraes et al., 1998;

McCormick et al., 2012). These tritrophic interactions involved in plant defence and the

intricate mechanisms (Mercke et al., 2004) behind them illustrate the complex role plant

secondary metabolites serve in plant-insect relations.

Plant volatiles have a variety of other functions in their environment. Plant volatiles do not

only reduce the amount of attacking herbivores (Kessler & Baldwin, 2001) through typical

plant-parasite/predator interactions but can also alert neighbouring plants (both conspecifics

and heterospecifics) of any immediate herbivore or pathogen danger (Shulaev et al., 1997).

Volatiles can induce the expression of defence genes in neighbouring plants (Arimura et al.,

2000) or prepare these plants to respond more efficiently to attacks in the future (Kessler et

al., 2006). Certain plant-emitted volatiles are also capable of adhering to neighbouring

heterospecific plants and subsequently affect their interactions with their environment

(Himanen et al., 2010).

9

In addition to the examples and literature mentioned previously, there have been countless

others which have demonstrated the use of plant-emitted volatiles as an indirect mechanism

where plants make use of the natural enemies of herbivores to serve as ‘bodyguards’

(Takabayashi et al., 1991; Van Poecke & Dicke, 2004). Although the effect herbivore-induced

plant volatiles have on attacking herbivores remains more or less unchallenged in the current

literature, while some agree plant fitness is hugely affected by the ecological functions of their

secondary metabolites (Hopkins & Huner, 2009; Lazar et al., 2003); others remain unclear

whether the release of these volatiles have any net fitness gain for the plant. Therefore, more

studies into the evolutionary ecology of herbivore induced volatiles are required in order to

fully grasp these mechanisms (Dicke & Baldwin, 2010).

Secondary Compounds in Ferns

This review touches briefly upon a small number of studies that provide evidence of secondary

metabolites used as successful defence strategies in higher plant groups. Unfortunately, I have

yet to come across publications that directly investigate the effect secondary metabolites have

on the defensive capabilities in the fern taxa after extensively probing the literature. The most

recent review on the potential biological functions of secondary metabolites in ferns by Vetter

(2018) provides a comprehensive summary on the chemical and biochemical aspects and uses

but only briefly addresses their role in plant defence. The production of cyanogenic glycosides

to work against biological attackers and the effectiveness of tannin molecules as deterrence to

phytophagous insects were reported in this review.

Other reviews have investigated fern phytochemicals such as that of Cao et al. (2017) in which

they provide an extensive summary of chemicals found in an array of fern species throughout

the world. Though they note evidence suggesting potent antimicrobial activity in multiple

studies and the toxicity of bracken ferns from sesquiterpene glycosides, their conclusions and

discussions concerning fern chemicals revolve around the potential medicinal and

pharmaceutical applications. However, Cao et al. (2017) also briefly report a case were

methanol fern extracts were successful as an insecticide but do not elaborate further.

Similar studies have shown evidence which suggests that secondary metabolites of fern

species Lygodium venustum having antibiotic uses in chemotherapy though nothing has been

investigated into their effectiveness against plant pathogens (Morais-Braga et al., 2012).

Although not directly attributed to secondary compounds, nectar secretion of fern species

Pleopeltis crassinervata were associated with lower levels of herbivore damage (Koptur et al.,

2013). It is obvious from gleaning through the literature that recent research regarding fern

10

phytochemicals are led by medical and pharmaceutical journals mainly as novel treatments or

approaches to microbial studies (Khan & Ullah, 2018; Nath et al., 2018; Ullah et al., 2018).

Older research such as that by Soeder (1985) only provides a comprehensive survey of fern

constituents and does not give any insight as to their ecological importance.

Compared to non-volatile secondary compounds, there is noticeably more literature directly

concerning fern-emitted volatile organic compounds. Although there are some which do not

investigate the ecological importance of these volatiles (Froissard et al., 2011), there are a

handful which investigate the relationship between these volatiles and herbivory. Surprisingly,

there is evidence (Imbiscuso et al., 2009; Kessler et al., 2015) that certain fern species are able

to release herbivore-induced volatiles, however, both studies did not directly measure the

effect these volatiles had on the herbivores and herbivore activity.

Imbiscuso et al. (2009) observed an ‘oxidative burst’ in the fern species Pteris vittata and the

subsequent emission of volatile terpenoids following herbivore damage which is a trend many

other higher plants express upon herbivory. Similarly, Kessler et al. (2015) in their investigation

of six related species of fern; Melpomene firma, Melpomene wolfii, Alansmia laxa, Cochlidium

serrulatum, Lellingeria subsessilis, and Mycopteris taxifolia, reported compounds that were

well known among angiosperms as herbivore deterrents, particularly the mono- and

sesquiterpenes.

This is not to say there is no evidence against the use of volatiles as defence mechanisms in

ferns. Radhika et al. (2012) tested the volatile emission following herbivory of two different

species (one generalist, one specialist) on the bracken fern Pteridium aquilinum and concluded

that the jasmonic acid levels in both species were not sufficient enough to elicit volatile

emissions from the fern. However, it is important to know that the current amount literature

concerning this topic is insufficient and inadequate to make solid conclusions about the

defence strategies in ferns and thus further research into the ecology of fern volatiles is

needed.

11

Fern Insect Interactions

Most major groups of vascular plants, to some degree, are used by phytophagous insects as a

source of food and ferns are no exception. However, there is relatively little information in the

literature on fern-insect interactions. The most recently relevant studies broadly discussing this

topic were written over three decades ago (Auerbach & Hendrix, 1980; Balick et al., 1978;

Cooper-Driver, 1978; Hendrix, 1980). Since these publications there has been a paucity of

studies building onto their work and only very few publications on specific fern-arthropod

interactions over this time.

It is a widespread belief that ferns have few insect attackers. Schneider (1892) was the first to

describe the relatively poor number of insects feeding on ferns compared to flowering plant

and conifers. The phenomenon was reiterated again by Brues (1920) and challenged soon after

by Swezey (1922) through his observations in the Hawaiian Islands though his conclusions

were later called into caution (Hendrix, 1980). While over the years other authors repeatedly

reported poor herbivory on ferns (Hoo & Fraenkel, 1964; Kaplanis et al., 1967), these reports

were generally based on anecdotal evidence. Balick et al. (1978) was one of the first to

systematically survey the literature to solve the long-held observation as well as the earliest to

suggest a possible coevolution between arthropod and ferns prior to and after the radiation of

angiosperms. Hendrix (1980) conducted a similar survey of the literature and additionally

compared this to Balick et al. (1978) and concluded that the total number of insects that

utilized ferns as a food source was significantly fewer than expected but suggested that the

amount of insects recorded to have fed on ferns were not a representative sample of

phytophagous insects as a whole thus emphasising the need for more studies. Hendrix (1980)

was also one of the first to highlight the potential importance of chemical defences and

ecological characteristics in fern-insect interactions.

Flaws regarding the age-old generalization that only few arthropods feed on ferns were

pointed out by Auerbach and Hendrix (1980) suggesting comparisons to angiosperms were

invalid due to biases involving plant growth form and unequal sampling efforts and reiterated

again by Hendrix and Marquis (1983) and by Mehltreter and Tolome (2003) in observations on

herbivore damage in tropical ferns, which are more diverse than those in the temperate

regions of the Northern hemisphere, where the majority of reports originated.

12

Studies have shown that ferns have evolved adaptations in response to predators, such as the

release of nectar from the fronds to recruit ants (Tempel, 1983), the relation between fern-

feeding moths and fern phylogenies (Weintraub et al., 1995), and changes in the sporing

season to avoid predation by spore feeders (Sawamura et al., 2009).

There is also ample fossil evidence suggesting a long history of interactions between insects

and ferns. Examples include the use of insect vectors for seed fern pollen dispersal in the

Upper Carboniferous (Scott & Taylor, 1983), pteridophyte spore predation all throughout the

Palaeozoic (Scott et al., 1985), and direct defence mechanisms against herbivory by a seeded

fern in the Late Carboniferous (Krings et al., 2003). Together, this evidence supports a

standing relation between insects and ferns and prompts for further investigation.

Methods for Volatile Collection and Analysis

Recently, interest into the ecology and biochemistry of plant volatile organic compounds has

increased, leading to the development of a variety of techniques and systems for the collection

and analysis of volatiles (Linskens & Jackson, 1997). Through the design of sensitive and

relatively inexpensive bench-top instruments for gas chromatography-mass spectrometry (GC-

MS), volatile analysis over the last couple decades has improved quite significantly.

Traditionally, methods such as steam distillation or solvent extractions were used (Veith &

Kiwus, 1977) to acquire the volatile compositions of living plants, however, with the rise in

developed headspace analysis techniques providing more representative volatile profiles, the

former methods of analyses are, although still practiced (Ormeno et al., 2011), less used in

modern volatile analyses.

The methods and techniques discussed in this section will primarily overview the practical

approaches to volatile analyses published by Tholl et al. (2006). This is because it is currently

the latest review regarding the practices involved with plant volatile analyses. Thus, if further

detail is required, it is worthwhile reading this publication as each technique is described in

more detail there.

There is no single, widely accepted method for acquiring plant volatiles but rather, the most

suitable system and techniques that are required are usually dependent on the plant

material/organ and biological question being investigated (Tholl et al., 2006). For example,

whether analyses of volatiles are done on plants under laboratory or field conditions will result

in completely different sampling techniques and use of instrumentation, each having their

respective advantages and disadvantages. Field systems will therefore be more simplified and

13

have been modified for portable volatile collections, whereas laboratory systems allow for

more precision and manipulation using intricate set-ups and additional devices to control for

other variables. Whether the experiment requires a ‘snapshot’ of the volatile profile released

or whether developmental or stress related changes need to be measured must also be

considered during experimental design (Tholl et al., 2006).

Solvent extraction is one of the oldest, more destructive methods of volatile sampling

(Harborne, 1998) but recently, the investigation into the airspace (headspace) surrounding

above-ground plant material, is being adopted by many studies (Bicchi et al., 2008; Fan &

Almirall, 2014; Ha et al., 2014) due to their non-destructive and more realistic volatile profiles.

There are two main headspace sampling techniques commonly used throughout the literature:

static and dynamic headspace sampling, each with their own strengths and limitations (Tholl et

al., 2006).

Analysis of the static headspace involves enclosing the entire plant (or parts of it) into a

container and subsequently extract volatiles using an adsorbent material. This technique does

not circulate air within the chamber, thus static, disallowing impurities from a continuous

airstream mixing in. The recent development of solid phase microextraction in this sort of

system allows for the sampling of volatiles at the ‘parts per billion’ level to become practical

and efficient (Tu et al., 2014). In contrast, dynamic headspace sampling uses a continuous air

stream and is the most frequently used technique in volatile analysis. There are two systems

associated with this sampling technique: closed-loop stripping and ‘push’/’pull’ systems (Tholl

et al., 2006). Closed-loop systems circulate the air in closed chambers whereas ‘push’/’pull’

systems use a constant flow of air taken up from the outside and leaves the system with the

volatiles in tow.

These sampling methods trap plant volatile organic compounds on adsorbent material and are

then routinely analysed by the standard technique of Gas-Chromatography (GC). For this

review, it is not practical to discuss the standard protocols used in GC analysis but there are

publications within the literature (Dewulf et al., 2002) who have. For GC analysis, plant

volatiles are either injected into the heated injector as solvent extracts or desorbed from the

adsorbent via a thermal desorption tube (Tholl et al., 2006). Mass spectrometry (MS) detectors

are the most widely used detectors in GC analysis of plant volatiles and are crucial in obtaining

ion chromatograms. Once compound profiles have been thoroughly analysed using the GC-MS,

identification of these compounds are deduced by comparing the results to the mass spectral

data from popular databases (e.g. NIST MS). Correct identification of these compounds can

14

simply be done using retention indices or similarity comparisons with library mass spectral

data (Tholl et al., 2006).

Research Objectives

Much of what we know regarding the regulation and ecological function of volatile organic

compounds comes from studies of higher plants, however, very little is known about their

function in lower plant groups such as ferns. This research will help elucidate ecological

interactions involving native ferns and their associated arthropod communities as well as give

insight into the evolution of anti-herbivore defence mechanisms in plants.

The purpose of this research is to characterise the volatile emissions of six New Zealand ferns

and to investigate whether these ferns emit volatile organic compounds as a response to

damage and whether this emission is related to other forms of direct defence (i.e. phenolics). I

further elaborate on whether they can differentiate between herbivore damage,

phytohormone treatment and mechanical wounding. Overall, this study aims to increase the

information on the subject and serve as a foundation for future studies in this topic.

The thesis addresses the following objectives:

1) To characterize the volatile emission of six native fern species

2) To investigate changes in volatile emission of six native fern species under three

treatments: herbivore damage, phytohormone treatment and mechanical wounding

3) To compare the volatile emissions between treatments and establish if qualitative

or quantitative differences occur

4) To establish volatile emission correlates with mode of growth (tree, epiphytic or

shrub) or other potential defence traits such as phenolics

Chapter 2 : Materials and Methods

19

Study Site and Fern Species

The ferns used in this study were found naturally growing in two vegetative areas within the

Manawatu Massey University campus (see Fig.2-1) during the last two months of 2016 and

first two months of 2017. The following six fern species were used for volatile analyses: silver

tree-fern (Cyathea dealbata); black tree-fern (Cyathea medullaris); rough tree-fern (Dicksonia

squarrosa); hound's tongue-fern (Microsorum pustulatum); hen and chicken fern (Asplenium

bulbiferum); and the shining spleenwort (Asplenium oblongifolium) (see Fig.2-2).

Figure 2-1. The location of vegetative areas (red circles) within Massey University (Google Earth).

Only individuals with relatively small damage to their fronds were selected for treatments,

minor lacerations and sunburn to fronds were ignored. Ferns in positions where high

environmental stress may potentially occur, such as high exposure to sunlight or wind, were

avoided. Vegetative material used for volatile collections was undamaged and free of

invertebrate activity.

20

Figure 2-2. Native fern species used for volatile analysis; (a) Dicksonia squarrosa, (b) Microsorum pustulatum, (c) Asplenium bulbiferum, (d) Asplenium oblongifolium, (e) Cyathea dealbata, (f) Cyathea

medullaris.

Solvent Extraction

To characterise the volatile compounds of the six species, a solvent extraction method was

initially used. For this purpose, fresh aerial parts of ferns were collected from the same

vegetative areas of Massey University mentioned previously. Parts from the ferns species: C.

dealbata; C. medullaris; D. squarrosa; M. pustulatum; A. bulbiferum; and A. oblongifolium were

used for this analysis. Fresh plant material from over five individuals of each species were cut

into small pieces and accurately weighed to 3 grams. The samples were extracted using a 30

mL solution of high grade hexane (CAS 110-5-3, ≥99%, Sigma Aldrich, St Louis, MO, USA) and a

known concentration of nonyl acetate (C11H22O2) (10ng/mL) in a sealed container (Fig.2-3) for

72 hours in a climate-controlled room (22°C). During this extraction process the samples were

systematically shaken every 24 hours to increase the efficiency of extraction. Extracts were

then filtered through 70mm glass microfiber filters and again using 0.2μm syringe filters before

GC-MS analysis. The experiment was conducted in triplicate.

21

Figure 2-3. Examples of solvent extraction process: (a) initial filtering process using 70mm glass microfiber filter, (b) sealed containers containing fern material submerged in solvent/internal standard

solution.

Phytohormone and Mechanical Damage

To test the effect of different treatments of volatile emission, a dynamic headspace volatile

collection was carried out on site, using the same species described above. This method was

selected since it was non-destructive allowing to investigate changes over time. I submitted

each fern species to the following treatments 1) mechanical wounding; 2) mechanical

wounding plus phytohormone (jasmonic acid), and 3) control (no applied damage). Jasmonic

acid was used since it is the main phytohormone involved in plant anti-herbivore responses

and it is known to elicit herbivore-induced volatiles (Hopke et al., 1994).

Each treatment consisted of 5 replicates per fern species and had a total volatile collection

time of 3 hours during the same time of day (10:30-13:30) for each measurement. Mechanical

wounding of the plant material was conducted via standardised (30cm) linear tissue scarring

with a razor blade on the adaxial surface of the fronds. Application of the phytohormone

jasmonic acid was done using a spray bottle containing a 10mg/L solution of the compound

diluted in MiliQ H2O. Proceeding the tissue scarring of the plant material, 1mL of the solution

was sprayed evenly across the artificially damaged portions of the fronds prior to headspace

collection.

22

For VOC collection, the fronds were bagged using unused commercial oven bags enclosing

roughly 20-40 cm of the fronds (see. Fig.2-4). Collection of volatiles were conducted in the field

using a portable volatile assay system (PVAS22, Volatile Assay Systems, Rensselaer, NY, USA)

coupled with Teflon and silicon tubing for dynamic (push-pull airflow) headspace collections. A

90 ‘push’ and 80 ‘pull’ airflow scheme was set to create positive pressure within the system.

Volatile organic compounds were collected using an adsorbent (HayeSep Q, Volatile Assay

Systems, Rensselaer, NY, USA) filter at the ‘pull’ portion of the system. All collections were

conducted on dry, sunny weather conditions to minimise environmental influence on volatile

release. Sampled plant material subsequently had their fresh weight measured immediately

following volatile collection and dry weight after a minimum of 72 hours of drying in an 80°C

oven.

Figure 2-4. Example of a field experimental setup of the dynamic volatile headspace collection system using a PVAS22 unit on Dicksonia squarrosa.

23

The HayeSep Q filters were eluted using 200μL of high grade pentane (CAS, 109-66-0, ≥99%,

Thermofisher Acros Organics, Geel, Belgium) and a 1ng/mL nonyl acetate internal standard

solution. A 10ng/mL concentration of the internal standard was used for 1 replicate in the

control treatment for optimization purposes. Volatile solutions were then stored in -80°C

temperatures and briefly thawed prior to GC-MS analysis.

Polyphenol & Chlorophyll Collection

Analysis of polyphenol and chlorophyll content were conducted using a Dualex Scientific+™

(Force-A, Paris, France) sensor. A total of 10 measurements were randomly collected from

undamaged leaf material across each replicate of fern species. The embedded datalogger was

used to acquire the flavonol, anthocyanin, chlorophyll and nitrogen balance measurements.

Herbivore Damage

Assessment of the effect herbivore damage have on fern volatile emissions were tested on the

four fern species: C. dealbata; D. squarrosa; A. bulbiferum; and A. oblongifolium using the

Wellington tree weta (Hemideina crassidens) and passion vine hopper (Scolypopa australis) as

herbivores. These species were selected based on field observations, where they were found

to feed on ferns. Preliminary herbivore damage tests were conducted to estimate the damage

extent and overall effectiveness of the H. crassidens as a fern herbivore (Fig.2-5). These trials

consisted of using undamaged fresh frond cuttings from a variety of ferns and leaving them in

ice-cream containers with a single weta over the course of 48 hours. Observations from these

trials assisted with the conception of the primary experiment.

Figure 2-5. Example of herbivore damage by H. crassidens on frond cuttings of C. medullaris in preliminary trials.

24

Due to the difficulty and limitations associated with conducting herbivory experiments in the

field, these trials were held using potted fern species acquired from Fronds NZ Ltd (Cambridge,

New Zealand). It was unclear as to the origin of each fern individual though Fronds NZ Ltd state

that they source the majority of their ferns from pine forest areas around the country and are

subsequently replanted at their nursery’s and grown in ideal conditions. A total of 12

individuals for each of the target fern species were used for the experiment. During the course

of this study these plants were kept in a nursery alongside other non-fern New Zealand native

plants and watered regularly. The herbivores used to elicit volatile responses were sourced

locally. The Wellington tree wetas (H. crassidens) were gathered from a nearby patch of native

bush (Bledisloe Park) while the passion vine hopper’s (S. australis) was collected from

vegetation all around the campus grounds.

H. crassidens were gathered six weeks prior to volatile collections and were fed a non-fern diet

consisting of leaves from the native tree mahoe (Melicytus ramiflorus) and cubes of carrot.

They were kept in a climate-controlled room (16°C) with a fixed light regime (07:00-19:00 light)

and released back into the native bush at the conclusion of the experiment. In contrast, S.

australis were collected prior to volatile measurements and released the following day in a

completely different area from the place of collection. A new batch of S. australis were

collected for each volatile measurement.

Figure 2-6. Example of a laboratory experimental setup of the dynamic volatile headspace collection system using a PVAS22 unit on D. squarrosa.

25

The primary experiment consisted of 2 treatments, damage by H. crassidens and S. australis,

coupled with a control for each of the four fern species. A total of four replicates were used for

each treatment with volatile measurements taking place before the application of the

herbivore (15:00-17:00), during herbivore application (17:00-19:00), and 14 hours following

the removal of the herbivore from the plant to test for delayed volatile release (09:00-11:00).

Collection times were determined to accommodate activity times of H. crassidens. Volatile

measurements were acquired similarly to that of the phytohormone experiment (see Fig. 2-6)

in a climate-controlled room (17°C) with a fixed light regime (08:00-20:00 light). Control

measurements consisted of volatile collections in the absence of herbivores, weta treatments

involved placing both a single male and female weta on the fronds, and passion vine hopper

treatments involved 6 individuals freshly gathered individuals placed on the fronds (Fig.2-7).

Figure 2-7. Examples of insects interacting with ferns: (a) S. australis resting on D. squarrosa, (b) H. crassidens consuming pinna leaves of D. squarrosa.

26

Following volatile collections of herbivore damage, insects were immediately removed from

the plant and kept in containers overnight and separately measured the following day to

identify non-plant emitted volatiles from the samples. This was repeated for each replicate in

each of the two treatments. Prior to each herbivore damage experiment H. crassidens were

starved for roughly 50 hours each time to ensure feeding of fern material and reduce the

likelihood of frass occurring.

Volatile samples contained in HayeSep Q filters were eluted using 200μL of high grade pentane

(CAS, 109-66-0, ≥99%, Thermofisher Acros Organics, Geel, Belgium) and analysed through a

GC-MS. Volatile solutions were then stored in -80°C temperatures and briefly thawed prior to

GC-MS analysis.

Gas Chromatography-Mass Spectre Analysis

Analyses for fond extracts, phytohormone treatment, mechanical damage, and herbivory

experiments were performed using a Shimadzu benchtop gas chromatography – mass

spectrometer system (GCMS-QP2010, Shimadzu Corp., Kyoto, Japan) equipped with a TG-5MS

capillary column (30 m x 0.25 mm x 0.25 μm, Thermo Fisher Scientific, Waltham, MA, USA).

The injection volume of each sample was 1 μL. Helium (99.999%) was used as the carrier gas at

a column flow-rate of 1 mL/min. Carrier gas conditions include pressure control mode of 53.5

kPa, total flow (14.0 mL/min), linear velocity (36.3 cm/sec), purge flow (3.0 mL/min and a split

ratio of 10.0. Injection port temperature was 230 °C with the following column temperature

program: 50 °C for 3 min, followed by an increase to 95 °C at a rate of 5 °C/min, an increase to

145 °C at a rate of 15 °C/min, an increase to 200 °C at a rate of 10 °C/min and maintenance at

270°C for 3 min. The MS settings are as follows: ion source temperature (200°C), interface

temperature (200°C), solvent cut time (2 min) with a total program time of 23.83 minutes.

Figure 2-8. Example of manual peak integration using ion chromatography.

27

Peaks in chromatograms of volatile and solvent profiles were manually integrated and

analysed (see Fig.2-8). The NIST05 MS library (National Institute of Standards and Technology,

Gaithersburg, MD, USA) was used for mass spectral analyses and compound identification

according to reference compounds in the database (see Fig.2-9). Compounds were identified

based off known retention times of external standards. Only compounds with high similarity

values (≥80) were reported, headspace and internal standard contamination (i.e. noise) were

excluded from the analysis. In cases where compounds showed low similarity values (<80) but

retention times corresponded with previous reports, the compound was considered for

analysis.

Figure 2-9. Example of compound identification comparing mass spectrum data from samples to target reference values within the NIST05 MS library.

28

Statistical Analyses

Extract constituents were quantified by comparing unknown peak areas with the internal

standard (nonyl acetate). The constituents for both the phytohormone experiment and the

solvent extracts were quantified in the same manner. Analysis of volatile constituents acquired

from the herbivore experiments were done on the basis of presence/absence due to the

scarcity of data. Ion chromatogram characteristics such as peak area, peak height, and

area/height ratio were transferred to Microsoft Excel spreadsheets from all datasets. Means

and standard errors for the emission rate of each compound in the phytohormone and solvent

extract experiments were calculated and tabulated for qualitative analysis.

Values concerning the phytohormone treatment are expressed in ng g-1 DW h-1 (nanograms per

gram of dry weight per hour). Content concerning the solvent extract experiment are

expressed as the ng g-1 FW (nanograms per gram of fresh weight). Calculation of average total

emission rates per treatment (C, JA, or PD) were completed using the collective volatile

emission rates for each of the five replicates within the corresponding treatment. Emission

rates between fern species and growth mode (whether tree, shrub or epiphyte) were

calculated using the total sum of volatiles released per species per replicate. Comparisons

between species and growth type solvent extract analyses were conducted using the average

of the total sum of concentrations per replicate of species and plotted with 95% confidence

intervals.

One-way analyses of variances (ANOVA) as well as Tukey Honest Significant Differences (HSD)

post-hoc tests were used for treatment comparisons. All statistical analyses were conducted

using the R statistical computing software (Version 3.4.2; R Core Team, 2013). Bar plots were

constructed using Minitab 17 Statistical Software (2010).

Chapter 3 : Results

33

Solvent Extraction

A total of 7 compounds were detected and identified from six fern species using the solvent

extraction method (Table. 3-1). The concentrations of constituents were lowest in M.

pustulatum and its compound list (3 total) was also much lower than the other fern species

investigated. By contrast, D. squarrosa contained the highest concentrations of compounds

and released the highest number of compounds along with A. oblongifolium (7 total).

Comparison of the total concentration for constituents extracted based on species indicated a

difference in values (F=477.4, p<0.01). Post-hoc analyses reveals significant differences

between all individuals with the exception of CD-AB and CM-AO (Fig.3-1a). Comparison of

concentrations according to growth method determined differences in groups (F=3.73, p=0.04)

although further post-hoc analyses indicate no statistically significant differences between any

of the groups (Fig.3-1b).

Figure 3-1. Comparisons of confidence intervals (95%) and mean values of the concentrations of fern extracts according to species (a) and method of growth (b).

34

Table 3-1. Average concentration of fern extract compounds from fronds of six species. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 FW. Note: n.d – no data available.

Compounds Asplenium oblongifolium

Asplenium bulbiferum

Cyathea dealbata

Cyathea medullaris

Dicksonia squarrosa

Microsorum pustulatum

Hexanal

0.13 ± 0.02 0.50 ± 0.05 0.47 ± 0.02 0.27 ± 0.03 2.03 ± 0.06 0.07*

2-Hexenal

0.26 ± 0.03 0.69 ± 0.03 2.00 ± 0.16 0.59 ± 0.12 6.83 ± 0.12 n.d

3-Hexen-1-ol, (Z)-

0.47 ± 0.01 n.d 0.80 ± 0.01 0.48 ± 0.06 0.54 ± 0.07 n.d

2-Hexen-1-ol, (Z)-

0.65 ± 0.06 0.58 ± 0.06 n.d n.d 1.06 ± 0.12 n.d

2-Octen-1-ol, (E)-

n.d n.d 0.06 ± 0.01 0.57* 0.15 ± 0.03 0.14 ± 0.03

1-Hexanol

0.82 ± 0.03 1.37 ± 0.08 n.d n.d 1.01 ± 0.23 n.d

2-Nonen-1-ol

0.13 ± 0.03 0.14 ± 0.01 0.30 ± 0.02 n.d n.d n.d

1,6-Octadien-3-ol, 3,7-dimethyl-, acetate

n.d n.d n.d n.d n.d 0.18 ± 0.03

35

Phytohormone and Mechanical Damage

According to GC-MS analysis of headspace samples: 9 volatile organic compounds were

detected within the control (Table. 3-2), 10 volatile compounds from the phytohormone

treatment (Table. 3-3), and 11 compounds from the mechanical damage treatment (Table. 3-

4). The species of fern found to emit the fewest number of volatile compounds was A.

bulbiferum (8 total), followed by C. dealbata (9 total). D. squarrosa was found to emit the

highest number of compounds (12 total). The compound 2-Octen-1-ol, (E) - was consistently

detected throughout all fern species across both treatments and control. It was also the most

prominently released volatile compound in the control and phytohormone treatments. The

following compounds; 1,6-Octadien-3-ol, 3,7-dimethyl-, and 3-Hexen-1-yl, acetate, (Z)- were

detected only once during the control treatments. 1S-.alpha.-Pinene was found in several fern

species but never recurred in non-control treatments. Volatile compounds 3-Hexen-1-ol, 3-

Hexenal, (Z)-, 2-Hexenal, (E)-, and 1-Octene, 3,7-dimethyl- were present only in non-control

treatments.

Figure 3-2. Comparison of total volatile emission rates for each treatment; control - no induced damage (C) and two treatments - mechanical damage (PD), and phytohormone treatment (JA).

25

50

75

C JA PDTreatment

Em

issi

on(n

g g-

1 D

W h

-1)

36

Table 3-2. Average emission and SE of volatile organic compounds from fronds of six fern species within the control treatment. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 DW h-1. Note: n.d – no data available.



Cyathea dealbata

Cyathea medullaris

Dicksonia squarrosa


(Z,Z)-.alpha.-Farnesene

n.d n.d n.d n.d n.d 24.23 ± 20.45

1,6-Octadien-3-ol, 3,7-dimethyl-

n.d n.d n.d 1.82* n.d n.d

1S-.alpha.-Pinene

n.d n.d n.d 8.57* 1.26* 24.58*

2-Octen-1-ol, (E)- 19.49 ± 6.23 31.64 ± 13.39 7.79 ± 3.49 4.49 ± 1.12 7.62 ± 0.39 102.35 ± 66.26

2-Pentanone, 4-hydroxy-4-methyl-

3.64* n.d 1.67* n.d 2.33* 18.05*

3-Hexen-1-yl, acetate, (Z)-

n.d n.d n.d n.d 6.39* n.d

3-Octanol 6.63 ± 4.83 15.27 ± 6.02 n.d 0.97* 1.09* n.d

3-Octanone 7.06 ± 1.88 7.59 ± 2.58 1.97* 2.99* n.d 16.61*

Copaene 5.65 ± 2.44 6.40 ± 1.27 3.34 n.d n.d 5.23*

37

Table 3-3. Average emission and SE of volatile organic compounds from fronds of six fern species within the phytohormone (jasmonic acid) treatment. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 DW h-1. Note: n.d – no data available.



Cyathea dealbata

Cyathea medullaris

Dicksonia squarrosa



n.d 6.86* n.d n.d 1.27* 29.26 ± 26.60

1-Hexanol

2.60* 6.61 ± 0.81 n.d n.d 13.00* n.d

1-Octene, 3,7-dimethyl- 3.61 ± 0.09 2.88 ± 0.49 3.77 ± 0.68 4.82 ± 1.46 4.79 ± 0.45 9.74 ± 2.84

2-Hexenal, (E)- 4.73 ± 0.2 12.50 ± 2.42 n.d 6.46 ± 0.1 12.41 ± 7.61 10.78 ± 7.83

2-Octen-1-ol, (E)- 24.23 ± 7.39 60.90 ± 12.92 32.12 ± 5.81 13.89 ± 4.81 20.40 ± 7.56 222.15 ± 51.49


n.d n.d n.d n.d n.d 13.23*

3-Hexen-1-ol 2.09 ± 0.43 n.d 11.92 ± 4.30 3.70 ± 0.94 15.63 ± 7.02 n.d

3-Octanol 4.41 ± 1.61 22.47 ± 6.87 9.36* n.d 17.63 ± 12.9 n.d

3-Octanone 3.91 ± 0.36 15.94 ± 4.59 4.41 ± 0.96 11.41* 2.32* 11.20 ± 4.06

Copaene 5.83 ± 1.51 13.93 ± 6.94 n.d n.d n.d 10.82 ± 3.32

38

Table 3-4. Average emission and SE of volatile organic compounds from fronds of six fern species within the mechanical damage only treatment. Values indicated with a ‘*’ represent data from a single replicate. Values are presented in ng g-1 DW h-1. Note: n.d – no data available.



Cyathea dealbata

Cyathea medullaris

Dicksonia squarrosa



4.57* 2.40* n.d n.d n.d 38.24*

1-Hexanol

5.36 ± 1.22 5.87 ± 0.67 n.d 3.97* 11.20 ± 2.97 21.32*

1-Octene, 3,7-dimethyl- 3.25 ± 0.98 1.73* 4.70 ± 0.415 6.02 ± 1.71 5.48* 6.62*

2-Hexenal, (E)- 3.02* 8.03 ± 1.09 7.35 ± 1.55 n.d 60.27 ± 20.10 56.34 ± 44.95

2-Octen-1-ol, (E)- 50.15 ± 18.89 64.85 ± 10.53 53.87 ± 18.24 23.15 ± 3.88 75.50 ± 45.44 322.99 ± 146.91


n.d n.d n.d 8.66* n.d n.d

3-Hexen-1-ol 7.24 ± 3.20 n.d 33.05 ± 16.30 7.86 ± 2.23 39.04 ± 23.16 20.64 ± 2.68

3-Hexenal, (Z)- 59.82 ± 22.99 30.07 ± 5.92 72.99 ± 37.09 31.81 ± 5.97 156.12 ± 57.84 172.79 ± 88.19

3-Octanol 13.81 ± 5.59 28.98 ± 5.70 n.d 2.23* 15.76 ± 6.76 n.d

3-Octanone 11.45 ± 2.37 13.43 ± 1.99 8.21 ± 2.06 5.77 ± 0.57 n.d 12.64 ± 1.41

Copaene 11.35 ± 3.87 14.16 ± 3.75 n.d n.d n.d 20.02 ± 7.12

39

Comparison of total emission according to treatment yielded no significant differences in

means (F=2.16, p=0.158, Fig.3-2). Across all fern species investigated the control treatment

averaged 18.72, 95% CI [5.42, 32] ng g-1 DW h-1, jasmonic acid application 21.94, 95% CI [17.8,

26.1] ng g-1 DW h-1, and mechanical damage 41.98, 95% CI [20, 64] ng g-1 DW h-1.

Comparison of total emissions between species showed no significant differences for the

mechanically damaged treatments (F=2.096, p=0.101) and control treatments (F=1.952,

p=0.123) (see.Fig.3-3). Differences only within the phytohormone treatment were detected

(F=9.156, p<0.01) with TukeyHSD tests indicating differences between species; M. pustulatum-

A. bulbiferum (p=0.04), M. pustulatum-A. oblongifolium (p<0.01), M. pustulatum-C. dealbata

(p<0.01), M. pustulatum-C. medullaris (p<0.01), and M. pustulatum-D. squarrosa (p<0.01).

Post-hoc analysis of emissions via treatments within species showed differences in; AB (F=4.98,

p=0.03) between PD-CT (p=0.03), CD (F=4.37, p=0.04) between PD-CT (p=0.03), CM (F=13.66,

p<0.01) between PD-CT (p<0.01) and PD-JA (p=0.02), and DS (F=5.03, p=0.03) between PD-CT

(p=0.03). There were no significant differences between treatments in AO (F=3.42, p=0.07) and

MP (F=1.82, p=0.2).

Figure 3-3. Comparison of total volatile emission rates for each treatment according to fern species. Control (CT) with two treatments - mechanical damage (PD), and phytohormone treatment (JA).

Species MPDSCMCDAOABPDJACTPDJACTPDJACTPDJACTPDJACTPDJACT

800

700

600

500

400

300

200

100

0

Tota

l Em

issi

on (n

g g-

1 D

W h

-1)

Bars are One Standard Error from the Mean

Individual standard deviations are used to calculate the intervals.

Interval Plot of Phytohormone Treatments by Species

40

Comparison of total emissions according to the growth method showed statistically significant

differences within control treatments (F=5.214, p=0.012), phytohormone treatments (F=19.22,

p<0.01), and mechanical damage treatments (F=3.42, p=0.048) (see Fig.3-4). Subsequent post-

hoc tests indicate differences with epiphyte-tree species in the control (p=0.01), with epiphyte-

tree (p<0.01) and epiphyte-shrub (p<0.01) species in the phytohormone treatment, and with

epiphyte-shrub (p=0.047) species in the mechanical damage treatment.

Post-hoc analysis of total emissions via treatments within growth modes showed differences

in; tree species (F=7.60, p<0.01) between PD-CT (p<0.01) and PD-JA (p=-0.01), and shrub

species (F=5.75, p<0.01) between PD-CT (p=0.01). There were no significant differences within

treatments for the epiphyte species (F=1.82, p=0.2).

Figure 3-4. Comparison of total volatile emission rates for each treatment according to fern growth mode. Control (CT) with two treatments - mechanical damage (PD), and phytohormone treatment (JA).

(Note: Tree = CD, CM and DS; Epiphyte = MP; Shrub = AB and AO).

Growth TreeShrubEpiphytePDJACTPDJACTPDJACT

800

700

600

500

400

300

200

100

0

Tota

l Em

issi

on (n

g g-

1 D

W h

-1)

Bars are One Standard Error from the Mean

Individual standard deviations are used to calculate the intervals.

Interval Plot of Phytohormone Treatments by Growth Mode

41

Polyphenol and Chlorophyll Analysis

Comparison of chlorophyll contents between species of ferns indicated a significant difference

in means (F=70.42, p<0.001, Fig.3-5a), post-hoc analysis shows difference between all species

with the exception of CD-DS and CM-MP. Subsequent analyses on flavonol content (F=73.41,

p<0.001, Fig.3-5b), anthocyanin content (F=6.91, p<0.001, Fig.3-5c), and the nitrogen balance

index (NBI) (F=34.71, p<0.001, Fig.3-5d) similarly indicated a significant difference of species

means regarding each phenolic. Post-hoc tests showed no differences in means of AB-MP, CD-

AO, CM-AO for flavonol and CM-AB, DS-AB, MP-AO, DS-CM for the NBI. Post hoc of

anthocyanin shows differences in means of only between MP-DS, DS-CM, DS-AB, and AO-AB

while other comparisons had no differences.

Figure 3-5. Comparisons of confidence intervals (95%) and mean values of chlorophyll content (a); flavonols (b); anthocyanins (c); and nitrogen balance index (d) of the six native fern species.

42

Analysis of other metabolic constituents according to method of growth resulted in no

significant differences in chlorophyll content (F=1.70, p=0.184, Fig.3-6a) and anthocyanins

(F=2.54, p=0.081, Fig.3-6c) but significant differences of values in all growth modes for

flavonols (F=69.56, p<0.001, Fig.3-6b) and the nitrogen balance index (F=28.44, p<0.001, Fig.3-

6d).

Figure 3-6. Comparisons of confidence intervals (95%) and mean values of chlorophyll content (a); flavonols (b); anthocyanins (c); and nitrogen balance index (d) of the three growth modes. (Note: Tree =

CD, CM and DS; Epiphyte = MP; Shrub = AB and AO).

43

Herbivore Damage

According to dynamic headspace GC-MS analysis; 7 volatile organic compounds (Table.3-5)

were detected over the course of this experiment. A. bulbiferum was found to have the most

number of volatile compounds emitted (7 total) in contrast to the rest which was observed to

emit a total of 4 compounds. GC-MS data concerning measurements acquired from the S.

australis - passion vine hopper (PVH) treatments were virtually non-existent with only A.

oblongifolium emitting enough to be detected on the GC-MS. Compounds (Z,Z)-.alpha.-

Farnesene, 3-Octanol, and Copaene were observed only on A. bulbiferum. Compounds 2-

Penten-1-ol, (E)- and 2-Hexene, (E)- were only detected during the application of wetas to fern

material. 2-Hexene, (E)- was detected in one instance 14 hours after herbivore removal.

In samples with volatile data spanning across the three time periods measured, peak area

during the herbivore application period were relatively smaller compared to that of

measurements acquired prior to and 14 hours after their application (see Fig.3-7)

Table 3-5. List of compounds detected from the herbivory experiments and corresponding treatments they were observed within. Control (CT), Weta (W) - H. crassidens, Passion Vine Hopper (PVH) – S.

australis. n.d – no data available.



Cyathea dealbata

Dicksonia squarrosa


n.d CT only n.d n.d

2-Hexene, (E)- W only W only W only W only

2-Octen-1-ol, (E)- CT/PVH/W CT/W CT/W CT/W

2-Penten-1-ol, (E)- W only W only W only W only

3-Octanol n.d CT n.d n.d

3-Octanone CT/PVH/W CT/W W only W only

Copaene n.d CT only n.d n.d

44

Figure 3-7. Example of three ion chromatograms of a control replicate for A. bulbiferum. (a) Chromatogram of volatile measurements prior to herbivore application period, (b) Chromatogram of volatile measurements during the herbivore application period, (c) Chromatogram of volatile measurements 14 hours after herbivore application

period. Note: Red lines indicate target volatile compounds.

Chapter 4 : General Discussion

49

Solvent Extraction vs. Headspace Collection

There have only been two studies published to date which have used solvent extraction to

analyse the metabolic constituents of ferns (Fons et al., 2010; Froissard et al., 2011). Another

study (Halarewicz & Szumny, 2010) analysed volatile oils extracted from the bracken fern

(Pteridium aquilinum sub. Aquilinum) using a similar method. Solvent extraction was also used

to investigate the VOC’s of six horsetails (Equisetum), a subclass of plants within the fern

lineage (Pryer et al., 2001), in a study which reported a sizable seventy-five VOC’s from these

plants (Fons et al., 2013). Although, much of my methods used for solvent extraction were

derived from Froissard et al. (2011), we obtained differing results. In their publication, they

reported roughly 30 compounds for each fern species analysed containing a range of aromatic

compounds, polyketides, monoterpenes and isoprenoid derivatives. In contrast, the highest

number of compounds detected in my samples was 7.

Froissard et al. (2011) investigated 6 ferns (Asplenium trichomanes, Dryopteris dilatate,

Polystichum setiferum, Gymnocarpium dryopteris, Pteridium aquilinum, and Phegopteris

connectilis). Upon comparison of constituent profiles between the two data sets, I found no

recurring compounds. Comparison with Fons et al. (2010) and their investigation of French

ferns (Adiantum capillus-veneris, Athyrium filix-femina, Blechnum spicant, Dryopteris filix-mas,

Oreopteris limbosperma) yielded equivalent results. I hypothesize that this may either be the

result of the physiological and/or environmental differences between species or the

differences in extraction methods. Froissard et al. (2011) had macerated and extracted fern

constituents for a period of one week whereas I had only extracted for a period of 72 hours

with scheduled shaking events. It is possible that due to the nature of ferns, longer extraction

periods and the crushing of plant cells may be necessary to obtain a higher number of

compounds, yet, crushing the leaves will damage the cell walls, releasing other compounds,

which are not normally found in the fern scent in nature.

Other studies have investigated the volatiles of ferns using headspace sampling techniques,

which are less invasive and do not require the destruction of the sample and are therefore

more similar to natural emissions (Imbiscuso et al. 2009; Kessler et al. 2015; Radhika et al.

2012). Comparison of data sets indicate recurring compounds (3-hexen-1-ol) with Imbiscuso et

al. (2012) and (a-pinene) with Kessler et al. (2015). A major reason for this difference in volatile

profiles is likely due to the fact that none of these studies measured the headspace directly in

the field but rather used dried (Kessler et al., 2015) samples or greenhouse propagated plants

(Imbiscuso et al., 2009; Radhika et al., 2012). As these conditions may not be representative of

50

natural stresses, volatile profiles were expected to differ. Despite being lab-based collections,

the number of compounds detected were similar to the ones reported in this study (Imbiscuso

et al., 2009; Radhika et al., 2012) with the exception of Kessler et al. (2015) identifying over 20

compounds from dried samples. As methodologies were different, it is very difficult to make

direct comparisons. Fern species used by Imbiscuso et al. (2009); Kessler et al. (2015); and

Radhika et al. (2012) are described in Chapter 1 of this thesis.

A direct comparison of both methods in this study revealed that headspace collections allowed

to recover a higher amount of compounds, although some compounds can be consistently

recovered by both methods. In terms of answering important ecological questions, in-field

headspace volatile analysis of plants would be more appropriate and provide a better output

of ecologically relevant compounds for further testing (i.e. in herbivory experiments). In

contrast, solvent extraction may be ideal for breaking down plant material in order to identify

key compounds in fern metabolism, but these may not necessarily be involved in fern defence.

As suggested by Froissard et al. (2011) solvent extraction can also be useful for describing likely

scents in plants for perfumery applications or even act as a compound check for GC-MS

analysis.

Phytohormone Treatment and Mechanical Damage

Jasmonic acid (JA) is a naturally occurring organic compound found in several plant species

with the function of regulating plant responses to biotic and abiotic stresses in addition to

plant growth and development (Delker et al.2006). Like with a similar study by Radhika et al.

(2012) I had expected the jasmonic acid to stimulate anti-pest defences within all ferns species

and thus increase its production and release of its volatile organic compounds, ultimately

however, these expectations were not met. Collectively, the fern species investigated did not

change their emission rates regardless of whether plant-defences were induced or not, but as

individual species, plants with epiphytic growths modes (M. pustulatum) appeared to produce

more volatile compounds compared to their more larger counterparts when exposed to JA. As

my results indicate, M. pustulatum has significantly higher concentrations of volatile emissions

compared to the other 5 fern species when damaged and stimulated by jasmonic acid. In

contrast, it was also identified as the species with the lowest concentration of volatile

constituents extracted. Though post-hoc analyses showed no significant differences between

growth modes, the uncertainty in this area may be clarified if the experiment were to be

repeated with larger samples sizes and a more representative array of species for each mode

of growth.

51

Studies investigating volatile emissions of epiphytic plants have yet to be published thus

currently, it is difficult to draw conclusions and support or deny any existing theories for this

observation. Based on my analysis, I suggest this explanation; epiphytes are under lower

habitat pressures save their need for a host for physical support, therefore the energy

investment to structures such as roots or bark are redirected into production of volatile

compounds for plant defences thus explaining why they may significantly release higher

concentrations. Alternatively, epiphytes are ideal habitats for animals, fungi and bacteria

(Everhart et al., 2009) thus it wouldn’t be uncommon for these plants to attract symbionts or

herbivorous predators towards the plant with their high emission concentrations. This may

also explain the fact that low concentrations of volatile constituents were extracted from this

species. It is possible that chemical reactions needed for synthesis of volatile compounds only

take place when cells on the surface are damaged. My conclusions concerning jasmonic acid

on epiphytic ferns are partly consistent with those of Radhika et al. (2012), indicating that

exogenously applied JA can trigger indirect defence responses (changes in VOC emission) in

some fern species.

Comparisons of volatile profiles between treatments however did yield interesting

information, 1,6-Octadien-3-ol, 3,7-dimethyl-, (Z)-3-Hexen-1-yl, acetate, and 1S-alpha-Pinene

were only ever detected in control treatments but never when mechanical damage was

applied. This suggests one of two scenarios; the first being that these compounds are passively

released by ferns but at incredibly low concentrations very difficult to detect and mechanical

damage dilutes these compounds even further on the GC-MS, whereas the second scenario

being that these compounds are released as a result of different stresses but due to artificial

wounding of plant organs, the plant subsequently prioritizes the new, more prominent

stressor, thus ceases or reduces the synthesis of ‘minor’ volatile compounds to more suitable

compounds that may effectively deal with the current threat. This hypothesis has yet to be

directly investigated within the literature however, studies by Geervliet et al. (1997) and Van

Den Boom et al. (2004) have expressed in their data similar observations where a small

handful of volatile compounds, non-identical to those in this study, were only observed in

undamaged treatments. Though not robust evidence for this hypothesis, it does warrant

investigation in future studies as it could be a potential discovery in plant defence research.

In relation to this theory, another interesting piece of information was revealed during

qualitative analysis, which was that the following compounds 3-Hexen-1-ol, (Z)-3-Hexenal, (E)-

2-Hexenal, and 1-Octene, 3,7-dimethyl- were only ever present in treated samples and absent

in the controls, usually, at relatively high concentrations compared to other compounds.

52

Likewise, with the studies mentioned above (Geervliet et al., 1997; Van Den Boom et al.,

2004), several compounds have also been reported to be present in only damaged samples.

This may support the second scenario I proposed but due to the paucity of research

concerning this, I cannot make any solid conclusions.

A brief look into the recent literature demonstrates that some of these compounds have

important roles in plant ecology; 3-Hexen-1-ol having shown to induce arthropod resistance

(Losvik et al, 2018), 3-Hexenal as a precursor to other important green-leaf volatiles (such as

(E)-2-Hexenal) (Kunishima et al., 2016), which has been shown to attract parasites (Vieira et al.,

2014). All these compounds are green leaf volatiles, often reported to mediate plant-

arthropod interactions in flowering plants (Light et al., 1993; Shiojiri et al., 2006; Whitman &

Eller, 1990). 1-Octene, 3,7-dimethyl- is an identified monoterpene but no direct plant-insect

function has been so far reported in the literature. However, other compounds belonging to

this group have been shown to play important roles in responses against abiotic stress and

natural enemy recruitment (Loreto et al., 1998; Wang et al., 2008; Yamasaki et al., 2007).

For the majority of fern species analysed, total volatile emissions differed significantly between

mechanical damage and control treatments. Similarly, when categorised according to method

of growth, these two treatments were also found to differ. Though this may not provide much

insight into the ecological aspect of fern volatiles it does provide evidence which shows, for

some fern species, that there are quantitative differences in the volatile emissions which

indicates that ferns respond to damage to cells, at least to some degree. These observations

are consistent with those of Van Den Boom et al. (2004) who also observed higher volatile

emissions in mechanical damage treatments compared to those collected from undamaged

leaves and other treatments for some compounds amongst a few higher plants. A possible

explanation as to why we observe this may be that because the plant responses are not

regulated by compounds such as JA, they may overcompensate for the immediate stress by

producing more than normal amounts of volatile compounds to “play it safe”, whereas when

responses are regulated, they would prioritise efficiency for energy spent thus producing the

fewer compounds for maximum efficiency.

53

Polyphenol and Chlorophyll Analyses

As depicted by Fig.3-5, there does not seem to be any pattern or trend in chlorophyll and

polyphenolic content amongst the fern genus. The similarities and differences in the means of

each species do not give us any indication as to the evolutionary development of these

compounds. Consultation of fern phylogenies (Pryer et al. 2004; Schneider et al., 2004)

suggests relatedness of Dicksonia and Cyathea genus and a more distantly related Asplenium

and subsequent correlation of these phylogenies to the differences inferred from data

suggests high variability in the production of these compounds or an environmentally driven

physiology. What is also noteworthy is the fact that there is no consistency with phenolic and

chlorophyll content between species of the same genus for instance, with the exception of

anthocyanins; chlorophyll, NBI and flavonols contents were significantly different amongst

species of the same genus (Asplenium and Cyathea). Although this isn’t sufficient evidence to

support any claim, it does raise the possibility that biosynthesis of volatile compounds in plants

could potentially be governed by habitat features rather than their genetic make-up.

Remarkably, we see a pattern in polyphenol production based on plant growth mode (Fig.3-6).

Leaf flavonols, in general, function to protect plants cells from UV, accumulates phenolics as

well as gives an indication as to the leaf light environment (Williams & Harborne, 1977).

Likewise, the nitrogen balance index (NBI) is an index used to estimate crop nitrogen nutrition

(Schröder et al. 2003). Given that tree fern species are more exposed to the sunlight

compared to epiphytes and shrub species it is understandable they invest more into the

production of leaf flavonols for extra UV protection. In contrast, epiphytes do not need to

invest in such compounds as they live mostly in the shade of their hosts. In addition to this,

some epiphytes serve as important nitrogen fixing plants in their habitat (Han et al. 2010) thus

may explain the relatively high nitrogen content in their leaves. In relation to the use of these

phenolics as a potential for defence against herbivory, there could be a relation between the

high nitrogen content and the high concentration of volatile compounds emitted in fern

epiphytes however these inferences also warrant future studies into this topic.

54

Herbivore Damage

Compared to volatile measurements acquired from other experiments in this thesis, GC-MS

analysis of volatiles from the herbivory experiment, were rather poor. The majority of

replicates across the four fern species investigated didn’t register any volatile compounds.

There are several likely explanations for this occurrence: the fact that measurements were

conducted in controlled conditions may have affected volatile release. These conditions may

not be reflective of the plants natural habitat ergo the plant had no reason to synthesize

volatiles. Likewise, greenhouse-grown plants may not respond equally to those growing in the

wild. Another explanation may be the fact that only 2 hours were used for volatile collection,

this time period may not have been sufficient to adsorb enough compounds to register in the

GC-MS. The environmental settings in the climate-controlled room (temperature, humidity,

photoperiod, etc.), may have not been optimal for VOC emissions (Gouinguené & Turlings,

2002). Comparison of volatile emissions under both field and laboratory conditions by Kigathi

et al. (2009) suggested that volatile release is likely to be influenced by additional biotic and

abiotic factors.

Interestingly, in control replicates where volatiles were registered in all or two of the three

volatile measurements, peak area of target volatile compounds appeared to have dropped

during the period of herbivore application only to increase again in the next measurement

(Fig.3-7). Although there isn’t any statistical evidence to support this explanation, it is possibly

due to the time of day used for measurements. First measurement occurred in the late

afternoon, followed by another in the early evening with the last in the following morning.

Volatile concentrations would seem to drop during the early evening and increase again the

following morning. This may indicate a temporal factor in volatile emissions in ferns however,

a dedicated study investigating this is required to confirm these observations. Other studies

investigating volatile emission dynamics have also found changes in emission during the course

of herbivory and with changes in photoperiod (McCormick et al., 2014).

Data regarding measurements associated with the passion vine hopper S. australis were

effectively none. Initially, this insect was chosen as a potential herbivore due to observations

of eggs and nymphs located on the underside of several fern species. In addition, this insect

has piercing and sucking mouth parts which serve as a contrast to the ripping and chewing

feeding method of H. crassidens. My results suggest-three possible scenarios; a) that the

damage done by them was insufficient to elicit a large response from the plant, b) that the

insects were simply not feeding during the volatile measurements either due to saturation of

55

food prior to collection or their peak activity times were inverse to the collection period, or c)

that they may not feed at all in some of the tested species. Either way, it is difficult to narrow

down the likely causes for these observations.

Lastly, the most important observation in this experiment is the emission of 2 compounds: (E)-

2-Penten-1-ol, and (E)-2-Hexene in weta herbivory treatments. The emission of these volatiles

corresponds with observations of fern material consumption by the weta and were detected

only during measurements when herbivores were present in the headspace. As no volatile

compounds were detected when insects were separately analysed or in any of the controls

these two compounds can be considered to have been herbivore induced. (E)-2-Penten-1-ol

has been recorded to be found only in herbivore treatments in the literature (Geervliet et al.,

1997; Van Den Boom et al., 2004) however, I have yet to find a study documenting its

effectiveness in plant-insect interactions. (E)-2-hexene is a green leaf volatile, green leaf

volatiles have been reported to mediate several plant arthropod interactions (Light et al.,

1993; Shiojiri et al., 2006; Whitman & Eller, 1990) but I have found no studies which report (E)-

2-hexene to be associated with herbivory. Further comparison with real standards is required

to validate the identity of these compounds and potentially use them in bioassays with natural

enemies of the herbivores.

Final Remarks

This is the first study, as far as I am aware, to investigate the volatile constituents of New

Zealand ferns under both field and laboratory conditions. As far as exploratory research goes, I

believe my work and the discussion above establishes a clear foundation on which future

studies can build upon. Through my experimentations, I have set a baseline on what to expect

when working with field-based trials with similar methodologies. Choosing the study species

for this thesis was partly due to their relative abundance in the natural environment and partly

due to their genetic and biological traits. I aimed to cover a range of aspects with what little

resources and time I was given to test the waters and, hopefully, establish certain priorities for

this topic. In addition, the two treatments I have carried out in the field (phytohormone,

mechanical), are common place in this line of work and the subsequent analyses, both

qualitative and quantitative analyses were somewhat successful in characterising the fern

volatiles I had initially intended to describe. Though I had expected to observe a larger variety

and higher quantity of volatiles when I first started, the results I found and conclusions I ended

up with leave me satisfied with the potential my research has on this issue.

56

Further observations into plant phenolics and fern growth mode provide a fresh perspective

on the evolution and perhaps underlying mechanisms behind the emission of fern volatiles and

even possibly fern-insect interactions. Though I failed to elucidate differences in volatile

emissions according to herbivore feeding methods due to a number of possible reasons (lack

of feeding by herbivores, greenhouse grown fern material, not optimal controlled conditions,

etc.), my observations can provide important information for future research designs. The

comparison between two collection methods was useful to determine the advantages and

limitations of each method, although there is surely much room for improvement in this area.

Chapter 5 : Conclusions and Recommendations

61

Conclusions

The primary aim of this thesis was to characterise the volatile emissions and volatile

constituents of six native fern species, to test their responses to herbivory, and investigate

whether the release of these compounds was related to the method of growth. I believe, with

my work through these sets of experiments, I have managed to fulfil the objectives I had set

out to investigate.

Analysis of volatile collections suggests that total volatile emissions did not differ significantly

between phytohormone induced and artificially induced damage however, there were

differences in the emissions between species within each treatment and between different

modes of growth. Polyphenol and chlorophyll content had no evident pattern between species

but suggests a trend based on the ferns method of growth. Despite, the relative paucity of

volatile data concerning my herbivory experiments, 2 compounds were correlated with direct

herbivory. Chemical compositions of fern material extracts were underwhelmingly informative.

Results from both extract and in-field phytohormone treatments show that solvent extraction

did not provide a representative chemical profile of the ferns. Although, solvent extraction

may serve as a potential check for particular compounds, it is not suitable for investigating

fern-insect interactions. This re-enforces the idea that in field measurements and collections

are crucial in determining fern-environment interactions. Lastly, qualitative analysis of volatile

organic compounds has identified 15 organic compounds which have been consistent

throughout my measurements and have been accurately identified with high similarity ratings,

additionally these compounds have been linked to plant physiology in the literature.

As my review of the literature demonstrates, there is an overwhelming gap in our knowledge

concerning the use of volatiles as defence mechanisms in ferns and other primitive plant

groups. Most, if not all the data acquired from these experiments are new and unchallenged.

As such, data from exploratory studies like mine may give insight into the evolution of anti-

herbivore defence mechanisms in plants by describing volatile constituents of one of the

oldest extant plant groups. Aside from the phylogenetic implications this research may have,

this also opens potential applications in the conservation of threatened fern species through

pest management using environmentally safe synthetic chemicals for biocontrol as well as the

novel idea for these volatile bouquets to be used in perfumery. The work from this study will

serve as a foundation for future projects in this growing field of research.

62

Recommendations for Future Work

Given that this field of research is relatively new, there are multiple areas for improvement

based on exploratory studies such as this and of those before. Particularly, the use of the latest

gas chromatography- mass spectrometry techniques and software are ideal for optimal

efficiency and accuracy in the quantitative and qualitative analysis this research entails. Data

processing can be overwhelming if investigating copious quantities without the use of

advanced software and technology. The equipment I was using was relatively outdated which

may have caused an overall decrease in the accuracy of my data - particularly the decade old

software and NIST library used for peak integration and compound identification. More recent

versions of these may have better resolutions or additional functions which may assist with

several functions relating to compound integration.

This research, despite the time and methodological constrains, opens the door for future

studies on fern chemical ecology. Future studies should include further comparisons between

diverse extraction methods to optimise the outputs, as well as the identification of other

potential herbivores, and new herbivory trials under natural conditions to elucidate the

responses of ferns. Multiple behavioural tests are also required to understand plant-herbivore

and plant-natural enemy interactions mediated by these volatiles. Additionally, a deeper look

into the fern´s genome and metabolome should reveal the signalling pathways involved in the

regulation of volatile emission and their evolutionary relevance.

Based on the results of my measurements concerning herbivory, it would be interesting to

investigate the temporal aspects of fern volatiles. As particular compounds, both plant and

artificial, have the potential to adhere to other plants, similar studies should be focused in

areas where such ‘noise’ is minimised or at least controlled to some degree. It would also be

crucial to dedicate entire studies solely on volatile profiles from different types of insect

herbivory as it would undoubtedly serve as an indispensable reference and guide for the

chemical bouquets we would expect from certain interactions and future research. Another

aspect in this topic worth investigating would be to study the responses of ferns to salicylic

acid, a plant phenolic known to induce flowering, cause allelopathy, and is essential to plant

disease resistance (Hopke et al., 1994; Raskin, 1992). On a final note, I would recommend and

welcome any interest into fern chemical ecology as it is an impoverished field of research, so

any contribution is more than appreciated. I would like to end with a quote from Cooper-

Driver (1978) which summarises the context of this thesis “is the apparent lack of feeding by

insects on ferns a fact or is it really due to lack of observations or even interest?”

65

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